Hybridization of target nucleic acid analyte to arrays of surface-bound nucleic acid sequence probes is an effective way to physically separate and detect defined nucleic acid sequences within a sample containing high sequence complexity. Array technology is particularly useful for monitoring cellular mRNA levels (gene expression), detecting and screening single mutation polymoiphisms, re-sequencing and potentially (de novo sequencing. The lower limit of detection of these array systems depends upon:
(1) the absolute number of target molecules in the analyte (either DNA or RNA); PA1 (2) the number of target molecules in the analyte which are able to hybridize with the surface-bound nucleic acid sequence probes; PA1 (3) the non-specific background binding; and PA1 (4) the density (or total number) of the detectable tags in the target molecule. PA1 (1) optimizing conventional techniques for direct incorporation of the tag, such as fluorescently-labeled nucleotides, into the target molecule; and PA1 (2) utilizing improved techniques that employ a secondary hybridization event in which a common signal amplification probe having a tag or dye directly attached is hybridized to the mRNA or cDNA at a high multiplicity, such as the method disclosed in U.S. Pat. No. 5,124,246 to Chiron Corporation. PA1 (1) hybridizing the first end of a bidirectional primer to a target nucleic acid sequence; PA1 (2) polymerizing at the hybridized first end of the bidirectional primer to form the complementary nucleic acid sequence to the target nucleic acid sequence; PA1 (3) hybridizing the second end of the bidirectional primer to a circular DNA template; and PA1 (4) polymerizing at the hybridized second end of the bidirectional primer using the circular DNA template in the presence of a multiplicity of signal amplification sequence units to form a complementary nucleic acid sequence to the target nucleic acid sequence to which is covalently attached a repeating signal amplification sequence,
There are a number of conventional methods to increase the absolute number of target molecules in the analyte. If the starting analyte is RNA, reverse transcription (RVT) followed by the polymerase chain reaction (PCR) or ligase chain reaction (LCR) may be used. If RNA is the desired final target, a combination of RVT/PCR followed by transcription with a phage RNA polymerase, such as T7 RNA polymerase, may be used. However, the exponential amplification obtained with RVT/PCR, for example, can significantly alter the final target distribution due to preferential amplification of certain mRNAs. Although this problem can be avoided by eliminating the PCR step and directly transcribing the reverse transcription cDNA product, transcription with T7 RNA polymerase routinely results in only about 100-fold target amplification. Unfortunately, this is unlikely to be sufficient to achieve the quantification and sensitivity required for gene expression systems for mRNA samples isolated from less than one million cells.
There are also a number of methods to increase the number of target molecules in an analyte that can effectively hybridize with the surface-bound nucleic acid probes. Reducing both the intramolecular structures of the target molecules and the probe molecules can facilitate the intermolecular hybridization process. When the application permits, this may be accomplished either by specifically designing the probes to have single-stranded character or by fragmenting the target molecules into shorter sequences, typically on the order of 50 to 100 nucleotides in length. Specific solutes, such as monovalent and divalent cations, polyamines and certain non-ionic detergents, may also facilitate hybridization by increasing the number of target molecules in the analyte that can effectively hybridize with the surface-bound nucleic acid probes.
Methods to decrease the non-specific background binding of the target directly depend upon the nature of the non-specific binding. Methods to decrease mis-hybridization of non-complementary targets to surface-bound probes generally involve decreasing mono- and divalent ion concentrations, addition of denaturants and increasing hybridization temperature. In addition to the latter two, addition of surfactants and carrier nucleic acid to the hybridization mixture may reduce non-specific binding of targets to the an-ay surface. Unfortunately, the tag moieties themselves can contribute to non-specific binding to both the allay surface and nucleic acid probes in ways that are not a priori apparent. Thus, having methodologies aimed at controlling and/or normalizing the chemical and structural environment of the tag molecules would be advantageous.
Methods to increase the density of the tag in the target molecules while suppressing dye-dye and base-dye quenching include:
While these optimizations and improvements can result in substantially higher target tag densities than can be currently achieved by direct incorporation of tags into the targets, the added complexity of the secondary hybridization step is likely to prohibit the use of highly denaturing conditions during the target hybridization step. This may limit or even prohibit the use of secondary hybridization schemes with array formats that require higher stringency conditions such as cDNA-based arrays.
Finally, little attention has focused on controlling the spacing of the tag molecules within the targets. Clearly, little control over this factor is possible when employing conventional random tag-incorporation methods. Having a method that facilitates the spacing of the tags in a defined three-dimensional configuration, or at least at a defined distance from one another, has a number of advantages. For example, in the case where the tags are fluorescently-labeled dye molecules, defined spacing can significantly reduce any potential dye-dye or nucleotide-base quenching of the dye molecules. Reducing dye quenching and hence increasing their quantum efficiency effectively increases the amount of signal that can be detected from a given target molecule.
The methods and kits of the present invention solve many of the problems attendant with conventional techniques of improving the lower limit of detection of array systems. The methods and kits of the present invention involve the direct covalent attachment of a repeating signal amplification sequence containing a spatially-defined tag onto the target molecule using rolling circle replication employing a bidirectional primer.
The mechanism of rolling circle replication is known in the art. For example, PCT/US96/18812 discloses rolling circle replication for amplifying the amount of a target oligonucleotide. The replication reaction can be performed using tagged nucleotides to incorporate a detectable tag into the amplified target. PCT/US96/18812 also discloses using rolling circle replication in combination with an array for multiple testing of samples in parallel. In the examples of PCT/US96/18812, a ligase is used to circularize the amplification product, i.e., a linear amplification product is not hybridized. The application does not disclose using rolling circle replication for amplifying the signal produced by the target nucleic acid sequence.
The rolling circle mechanism is also described, for example, by A. Kornberg and T. A. Baker (editors), DNA Replication, W. H. Freeman Publishing (1 992), page 113, wherein certain plant viroids and virusoids use a rolling circle mechanism to replicate their circular RNA genomes, and by A. Fire and S-Q Xu, Proc. Nat. Acad. Scie. USA 92, 4642-4645 (1995) and Liu et al., J. Am. Chem. Soc. 118, 1587-1594 (1996), wherein bacterial and phage DNA polymerases utilize small circular DNA (between 26 and 74 nucleotides long) as replication templates for synthesizing DNA products having a repeated sequence up to 12,000 nucleotides in length. In these references, the rolling circle mechanism is used in combination with DNA cleavage strategies to synthesize practical quantities of short, defined DNA oligonucleotides.
The use of bidirectional primers is also known in the art. For example, PCT/US96/05480 discloses a bidirectional primer for polymerase chain reaction amplification wherein the bidirectional primers are designed to be complementary to each end of the same target. In contrast, the methods and kits of the invention employ a bidirectional primer wherein a first end is complementary to a target nucleic acid sequence and a second end is complementary to a circular DNA template.